Electrophoresis is a process in which macromolecules are separated on the basis of their charge-to-mass ratios by forcing them to move through a carrier medium, such as a porous gel, by means of a voltage gradient applied across the gel. Molecules having uniform charge-to-mass ratios, such as DNA and RNA, are differentiated on the basis of size.
Gel electrophoresis is a technique frequently used for separation with proteins and nucleic acids. In traditional methodology, a gel is cast as a thin slab between plates (for vertical runs) or alternatively poured and cast onto a flat bed (for horizontal runs) and positioned between buffer/electrode compartments in an electrophoresis apparatus. An appropriate buffer solution is added to the buffer/electrode compartments of the apparatus, and a small amount of sample solution is then carefully pipetted into precast notches on top of the gel. Usually glycerol and a "tracking" dye are contained within the notches, or added to the sample solution. An electric current is then applied across the gel until the tracking dye band has migrated through the length of the slab. The electric current is discontinued, and the gel is removed from the apparatus. At this point the gel is analyzed to determine the relative migration of the sample molecules. The gel may be stained, for example, with a dye that binds to proteins or nucleic acids, and the gel may then be dried or preserved, or a photograph taken, thus rendering a permanent visual record of the size distribution of the molecules in the original sample solution.
Electrophoresis gels typically are cast to accommodate multiple samples in an elongated gel slab. The gel slab is usually formed by inserting a comb device into nonsolidified gel medium. Usually the comb device has a plurality of equally spaced protrusions which resemble the teeth of a hair comb. The gel medium is then allowed to "gel", or harden, and the comb is removed resulting in the formation of sample-receiving wells within the upper portion of the gel slab.
Gel electrophoresis run in the conventional manner, as described above, is inherently inefficient. Typically, an operator must load the wells of the gel slab manually with a hand-held pipette. Samples must then be run immediately to prevent diffusion of the samples into the buffer. Therefore, loaded samples may not be stored, and thus, an operator must run tests immediately rather than when convenient. Also, the number of samples per gel made in the conventional manner is limited in order to provide a target well large enough to be loaded accurately.
Roger C. Wiggins in an article entitled "Agarose Drop Method for Loading Thin Polyacrylamide Gels", Analytical Biochemistry 126, pp. 422-424 (1982), describes a technique for gel electrophoresis where molten agarose is added to a crystalline sample and allowed to solidify. Each sample is then separated by cutting the backing on which it is placed, and the sample is slid onto a glass back plate of a horizontal gel mold and placed in contact with a polymerized stacking gel. The disadvantages of the Wiggins' technique are that the process is more labor intensive than simple well loading, and only has utility for very small samples sizes. The technology does not lend itself to automation thus limiting the number of samples that could be analyzed in a period of time. The technology is also limited in that the resulting band profile is a function of the size and shape of the sample drop, leading to lane patterns that are not reproducible.
Automation has been attempted in efforts to increase sample throughput in electrophoresis processes. For example, in a development program for automating gel loading at Beckman Instruments, Inc., a programmable pipetting machine, the Biomek Workstation, has been developed. Each individual precast gel is mounted on the Biomek, electrophoresis samples are loaded by the instrument, and then the gel is removed. The disadvantage of this system is that only a single gel can be loaded at a time. Also, because of the limits of pipetting accuracy, sample loading wells have to be quite large to be accessed accurately by the robotic arm. It is difficult to accurately deliver the Biomek pipet into a thin sample well, and thin wells are desirable because they determine band resolution in the gel. If the well is made thick enough to allow accurate pipetting, significant band resolution is lost. Another disadvantage of the Biomek method is that only one gel at a time can be loaded. Because of the above problems, and the lack of ease of operation, the Biomek method of automated gel loading has not been adapted widely by the research community.
A system for automated gel loading also eliminates pipetting error which occurs when samples are switched or mixed up during hand-held loading. Thus, multiple robotic pipetting not only hastens the loading procedure but also protects against application of wrong sample sequences.
In designing the sample holder and automatable gel loading process of the instant invention, Applicant recognized several constraints. First, the efficiency of an automated process would be improved by increasing the number of samples which could be loaded simultaneously on a sample loading machine. However, when the size and spacing of sample wells is decreased in an effort to provide a higher density of wells per gel, the pipetting machine must be increasingly more precise in order to successfully deliver samples to the smaller wells. Hence, one problem to overcome is how to increase the number of sample wells per gel while maintaining a target well size that can be hit by a robot arm on the pipetting machine. A second related constraint involves trying to maintain a very thin well in the dimension of the direction of sample electrophoresis to maintain high band resolution. A third constraint involves the need to design a sample loading system whereby the automated sample delivery device can efficiently load samples for more than one gel slab at a time. A fourth constraint is the need to provide a system of loading samples wherein the loaded samples may be stored for a length of time before they are electrophoresed through the gel. Sample diffusion and integrity must be controlled during storage.
Hence, a better, more efficient method of automatable gel electrophoresis is needed. It is an object of the present invention to solve the problems associated with conventional gel electrophoresis by providing a method of loading samples into a separate sample holder unconnected from the gel slab, wherein the samples are allowed to gel within individual wells within the sample holder and can therein be stored until electrophoresis. Another unique and beneficial feature of Applicant's gel loading system is that sample aliquots are delivered to the sample well chamber from a position parallel and not perpendicular to the electrophoretic axis, in other words, into the front of the sample well chamber rather than into the "top" of the sample well chamber, relative to the direction of the electrophoretic current as the well is viewed when in position for electrophoresis. Thus, the surface area available for delivery of the sample to the well is greatly increased compared to traditional horizontal loading schemes, where the sample is loaded into the top portion of the well chamber from an angle perpendicular to the electrophoretic axis.